This project is focused on developing curative gene therapies for primary immune deficiencies (PIDs) and primary immune regulatory disorders (PIRDs). The project also must include clinical studies to understand the basic defects and clinical problems affecting the patient groups for which we are developing gene therapies; and management strategies to keep them alive and well so they may benefit in the future from gene therapies. We must also understand the genetic defects causing the disorders; the mutations affecting function of those genes; and the molecular biology/physiology of the regulation, transcription and translation of those genes. We use a variety of cell lines, primary patient cells, and animal models to develop these gene treatments; and a variety of tools including integrating and non-integrating gene transfer vectors, as well as gene editing reagents/approaches; as well as methods to transiently correct function in mature immune cells by transfection with mRNA (See patient filed regarding mRNA treatments- Ref #3). To these ends, we developed and studied integrating self-inactivating lentivectors and are using them in ongoing clinical trials that are open in our program at the clinical center to treat X-linked chronic granulomatous disease ( NIH Protocol #15-I-0008; ClinicalTrials.gov NCT02234934; 3 patients treated at NIH, 9 patients at all the centers including the United Kingdom- ClinicalTrials.gov NCT01855685; Also see Abstract report of these 9 patients: Kohn DB, et al. 2018 Mol Ther 26(Suppl 1):157) and older children and young adults with X-linked severe combined immune deficiency (NIH Protocol #11-I-0007; ClinicalTrials.gov NCT01306019; 8 patients treated at NIH with early report on the first 5 patients, De Ravin et al. Sci. Transl. Med. 2016 8:335ra57) as well as contributing scientifically to the conduct of an ongoing clinical trial of lentivector gene therapy for newborn infants treated at St. Jude Childrens Research Hospital (ClinicalTrials.gov NCT01512888; preliminary abstract report of 7 patients; See abstract: Mamcarz E et al. 2017 Blood 130(Supp 1):523). We have also contributed scientifically (Ref # 14) to a new clinical trial of SIN lentivector gene therapy for children with Artemis deficient Severe Combined Immune Deficiency being conducted by our colleagues at the University of California San Francisco (ClinicalTrials.gov NCT03538899; 1 patient already treated). In the context of our program we have developed improved methods of gene editing using CRISP/Cas9 (CRISPR), Zinc Finger Nucleases (ZFNs), or TALENs to correct PIDs in cellular models of gene correction/mutation repair of primary immune deficiencies (Refs #s 2,5,11,17,18); developed and studied stem cell models that include both primary CD34+ hematopoietic stem cells (HSCs) obtained from patients and healthy volunteers or iPSC developed from patients and healthy volunteers and from animal models that may be used as targets for gene therapy (Refs #s 1,2,5,11,12,14,17,18); studies the regulation of genes we are trying to gene edit (Refs #11,18); developed model systems of specialized immune cell types such as microglia, neutrophils and monocytes from healthy volunteer and patient induced pluripotent stem cells (Refs #11,12,18); participated in the discovery of new clinical phenotypes in the p40phox deficient form of chronic granulomatous disease (Ref #19); studied immune system function, regulators and gene function (Refs # 1,2,5,11,12,14,15,17,18,19); assessed the outcomes of allogeneic transplants and other cellular therapies given to patients with PIDs that serve as important guides to understand the target goals of gene therapy (Refs # 8,13); conducted studies of carrier females of X-linked PIDs, where such studies may also inform the target goals for gene therapy (Ref # 9); and conducted observational and standard of care management studies of patients with PIDs of special interest, which for this project, in particular involves patients with the various genetic forms of chronic granulomatous disease (CGD) (Refs # 7,8,10,16,19,20). We also continue to study gene editing to correct the WHIM immunodeficiency defect with our collaborators in the Philip Murphy laboratory. Accomplishments by publication and patent in 2017-2018 period Reference #: 1. Arai et al: Developed novel method of bone marrow conditioning using anti-cKit Chimeric Antigen Receptor T cells introduced by retrovirus vector in a mouse model achieving high efficiency of donor engraftment. 2. De Ravin et al: Developed novel method of gene editing repair of X-CGD causing mutation in patient blood stem cells using the CRISPR/Cas 9 editing.. 3. De Ravin and Malech: New patent from Malech lab using mRNA to correct oxidase defect in CGD patent neutrophils that they may be used as treatment for infections. 4. Dveksler and Malech: New patent from Dveksler lab using pregnancy specific glycoproteins to induce immune tolerance. 5. Hong et al: Collaboration with Dunbar lab to gene edit Macaque non-human primate iPS cells to target a safe harbor site to express therapeutic proteins. 6. Jones et al: Generation of novel cell model system to assess Agonists of the Adenosine 2a receptor for development of treatments to induce tolerance for prevention/treatment of graft versus host disease. 7. Keller, Notarangelo and Malech: Review of current management of chronic granulomatous disease and development of gene therapy. 8. Marciano et al: Experience in the use of donor granulocyte transfusions for infection management in CGD; providing tbackground rationale for development of patent indicated in Ref #3. 9. Marciano et al: Correlates percent oxidase positive neutrophils in x-linked carriers of CGD with infection susceptibility providing necessary information to inform gene therapy targets. 10. Margolis et al: Provides important information regarding management of CGD patient care in the transition from childhood to adulthood. 11. Merling et al: Shows that gene editing correction of the GT deletion in the pseudogenes (NCF1B and 1C) of p47phox gene (NCF1) can resurrect full function and p47phox protein production fix oxidase defect in the neutrophils derived from corrected iPS cells, providing rational to develop mutation specific gene editing treatment. 12. Pandya et al: 1st demonstration of microglial cells generated from iPS cells, providing model for this cell type that could include gene editing. 13. Parta et al: Largest single center cohort of approach to allogeneic transplant for CGD demonstrating most importantly that transplant can cure CGD patients who have persistent life-threatening fungal infections. 14. Punwani et al: 1st demonstration of an effective lentivector to treat ARTEMIS SCID; vector now been brought forward to a clinical trial in the Cowan lab. 15. Sowriraian et al: Demonstrates p47phox phagocyte oxidase component regulates dendritic cell differentiation from monocyte derived macrophages. 16. Straughan et al: Current update of management of liver abscesses occurring in CGD patients. 17. Sweeney et al: CRISPR/Cas9 generation of the CGD genotype/phenotype in the nsg mouse that can accept human marrow xenografts, as a new important model for testing gene therapy. 18. Sweeney et al: 1st demonstration in human CGD patient derived iPS cell model that retention of the first intron of CYBB is essential for efficient cDNA mediated expression for gene editing correction. 19. Van de Geer at al: Contributed to collaborators demonstrating range of phenotypes in a large cohort of patients with p40phox deficient CGD. 20. Wingfield et al: Report from our surgical collaborators of largest series of neck dissection treatment experience for severe neck infections in CGD.